LOCUS       sequence-367269-        4247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockin of truncated NGFR to target gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4247)
  AUTHORS   Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, 
            Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JJ, Li 
            H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, 
            Eyquem J, Marson A
  TITLE     High-yield genome engineering in primary cells using a hybrid ssDNA 
            repair template and small-molecule cocktails.
  JOURNAL   Nat Biotechnol. 2022 Aug 25:10.1038/s41587-022-01418-8. doi: 
            10.1038/s41587-022-01418-8.
  PUBMED    36008610
REFERENCE   2  (bases 1 to 4247)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2022 Aug 25:10.1038/s41587-022-01418-8. doi: 
            10.1038/s41587-022-01418-8."
COMMENT     Sequence Label: pTR-342 pUC19-tNGFR-P2A-CD28 N
FEATURES             Location/Qualifiers
     source          1..4247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..57)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     157..176
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     370..392
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     384..401
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     385..401
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1631..1687
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2032..2048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2032..2048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2045..2067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2056..2072
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2080..2086,2087..2104,2105..2110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2125..2146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2263..2280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2434..3022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2514..2533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3193..3984,3985..4053))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3816..3835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4054..4158)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4226..4244
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
       61 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      121 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
      181 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
      241 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
      301 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
      361 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc
      421 ggggatttca agcacccctt ttacacaact cttgatatcc atcaataaag aataattccc
      481 ataagcccat catgtagtga ccgactattt ttcagtgaca aaaaaaaagt ctttaaaaat
      541 agaagtaaaa gtctaaagtc atcaaaacaa cgttatatcc tgtgtgaaat gctgcagtca
      601 ggatgccttg tggtttgagt gccttgatca tgtgccctaa ggggatggtg gcggtggtgg
      661 tggccgtgga tgacggagac tctcaggcct tggcaggtgc gtctttcagt tcccctcaca
      721 cttcgggttc ctcggggagg aggggctgga accctagccc atcgtcagga caaagatggg
      781 ggcaggtgcc actggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg
      841 ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc ctgtacacac acagcggtga
      901 gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag ccttgtggag ccaaccagac
      961 cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac gtggtgagcg cgaccgagcc
     1021 gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg tcggcgccat gcgtggaggc
     1081 cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag gatgagacga ctgggcgctg
     1141 cgaggcgtgc cgcgtgtgcg aggcgggctc gggcctcgtg ttctcctgcc aggacaagca
     1201 gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc gacgaggcca accacgtgga
     1261 cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc cagctccgcg agtgcacacg
     1321 ctgggccgac gccgagtgcg aggagatccc tggccgttgg attacacggt ccacaccccc
     1381 agagggctcg gacagcacag cccccagcac ccaggagcct gaggcacctc cagaacaaga
     1441 cctcatagcc agcacggtgg caggtgtggt gaccacagtg atgggcagct cccagcccgt
     1501 ggtgacccga ggcaccaccg acaacctcat ccctgtctat tgctccatcc tggctgctgt
     1561 ggttgtgggt cttgtggcct acatagcctt caagaggtgg aacagccgcg ccaagcgctc
     1621 gggttcgggt gccaccaact tcagcctgct gaagcaggcc ggcgacgtgg aggagaaccc
     1681 cggccccatg ctcagactgc tcttagctct caacttattc ccttcaattc aagtaacagg
     1741 taaacaatgt taatgtcttt ctttctgtaa atattttttg aggtcttcca attggcttag
     1801 tttattttaa atttctaaca atgtgtgaaa tttgaacatt tgaagtgtag ttttgctgta
     1861 atagggcaat gtgttatttt gaaaatcatt gattctcaga ctacaatata gtatcttaac
     1921 aaaagtccct tggaaataaa ttttctaagc tattagtttc tctagctatt agttgatagt
     1981 gtcccatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
     2041 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2101 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2161 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2221 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2281 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2341 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2401 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2461 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2521 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2581 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2641 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2701 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2761 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2821 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2881 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2941 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3001 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3061 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3121 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3181 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3241 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3301 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3361 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3421 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3481 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3541 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3601 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3661 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3721 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3781 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3841 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3901 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3961 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4021 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4081 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4141 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     4201 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccct
//
